This application claims the benefit of U.S. patent application Ser. No. 15/048,007, filed Feb. 19, 2016, U.S. patent application Ser. No. 13/767,314, filed Feb. 14, 2013, and U.S. Provisional Patent Application No. 61/600,070, filed on Feb. 17, 2012, all of which are hereby incorporated by reference in their entirety.
Ubiquitin is a small 76-amino acid protein that is the founding member of a family of posttranslational modifiers known as the ubiquitin-like proteins (Ubls). Ubls play key roles in controlling many biological processes including cell division, cell signaling and the immune response. Ubls are small proteins that are covalently attached to a lysine on a target protein via an isopeptide linkage with a C-terminal glycine of the ubl. The Ubl molecule alters the molecular surface of the target protein and can affect such properties as protein-protein interactions, enzymatic activity, stability and cellular localization of the target.
There are 8 known human Ubl activating enzymes (known as E1s) (Schulman, B. A., and J. W. Harper, 2009, Ubiquitin-like protein activation by E1 enzymes: the apex for downstream signalling pathways, Nat Rev Mol Cell Biol 10:319-331). Ubiquitin and other ubls are activated by a specific E1 enzyme which catalyzes the formation of an acyl-adenylate intermediate with the C-terminal glycine of the ubl. The activated ubl molecule is then transferred to the catalytic cysteine residue within the E1 enzyme through formation of a thioester bond intermediate. The E1-ubl intermediate and an E2 interact, resulting in a thioester exchange wherein the ubl is transferred to the active site cysteine of the E2. The ubl is then conjugated to the target protein, either directly or in conjunction with an E3 ligase, through isopeptide bond formation with the amino group of a lysine side chain in the target protein. Eukaryotic cells possess ˜35 ubiquitin E2 enzymes and >500 ubiquitin E3 enzymes. The E3 enzymes are the specificity factors of the ubiquitin pathway which mediate the selective targeting of specific cellular substrate proteins (Deshaies, R. J., and C. A. Joazeiro, 2009, RING domain E3 ubiquitin ligases, Annu Rev Biochem 78:399-434; Lipkowitz, S., and A. M. Weissman, 2011, RINGs of good and evil: RING finger ubiquitin ligases at the crossroads of tumour suppression and oncogenesis, Nat Rev Cancer 11:629-643; Rotin, D., and S. Kumar, 2009, Physiological functions of the HECT family of ubiquitin ligases, Nat Rev Mol Cell Biol 10:398-409).
Two E1 enzymes have been identified for ubiquitin, UAE (ubiquitin-activating enzyme) and UBA6 (Jin, J., et al., 2007, Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging, Nature 447:1135-1138). UAE is the E1 responsible for the majority of ubiquitin flux within the cell. UAE is capable of charging each of the approximately ˜35 E2 enzymes with the exception of Use1, which is the only E2 known to exclusively work with UBA6 (Jin et al., 2007). Inhibition of UAE is sufficient to dramatically impair the great majority of ubiquitin-dependent cellular processes (Ciechanover, A., et al., 1984, Ubiquitin dependence of selective protein degradation demonstrated in the mammalian cell cycle mutant ts85, Cell 37:57-66; Finley, D., A. et al., 1984, Thermolability of ubiquitin-activating enzyme from the mammalian cell cycle mutant ts85, Cell 37:43-55).
The cellular signals generated by ubiquitin are diverse. Ubiquitin can be attached to substrates as a single entity or as polyubiquitin polymers generated through isopeptide linkages between the C-terminus of one ubiquitin and one of the many lysines on a second ubiquitin. These varied modifications are translated into a variety of cellular signals. For example, conjugation of a lysine 48-linked polyubiquitin chain to a substrate protein is predominantly associated with targeting the protein for removal by the 26S proteasome. A single ubiquitin modification, or monoubiquination, typically affects protein localization and/or function. For example, monoubiquitination modulates the function of Histones 2a and 2b (Chandrasekharan, M. B., et al., 2010, Histone H2B ubiquitination and beyond: Regulation of nucleosome stability, chromatin dynamics and the trans-histone H3 methylation, Epigenetics 5:460-468), controls the nucleocytoplasmic shuttling of PTEN (Trotman, L. C., et al., 2007, Ubiquitination regulates PTEN nuclear import and tumor suppression, Cell 128:141-156), drives localization of the FANCD2 protein to sites of DNA damage (Gregory, R. C., et al., 2003, Regulation of the Fanconi anemia pathway by monoubiquitination, Semin Cancer Biol 13:77-82) and promotes the internalization and endosomal/lysosomal turnover of some cell surface receptors like EGFR (Mosesson, Y., and Y. Yarden, 2006, Monoubiquitylation: a recurrent theme in membrane protein transport. Isr Med Assoc J 8:233-237). Other forms of polyubiquitination include lysine 11, 29 and 63 chains which play various roles in the cell including the cell cycle, DNA repair and autophagy (Behrends, C., and J. W. Harper, 2011, Constructing and decoding unconventional ubiquitin chains, Nat Struct Mol Biol 18:520-528; Bennett, E. J., and J. W. Harper, 2008, DNA damage: ubiquitin marks the spot, Nat Struct Mol Biol 15:20-22; Komander, D., 2009, The emerging complexity of protein ubiquitination, Biochem Soc Trans 37:937-953).
UAE-initiated ubiquitin conjugation plays an important role in protein homeostasis, cell surface receptor trafficking, transcription factor turnover and cell cycle progression. Many of these processes are important for cancer cell survival and it is believed that tumor cells may have increased sensitivity to UAE inhibition as a result of their rapid growth rate, increased metabolic demands and oncogene fueled protein stress. Disruption of protein homeostasis is a validated therapeutic approach for the treatment of cancer. VELCADE®(bortezomib), disrupts cellular protein homeostasis and is approved for the treatment of multiple myeloma and mantle cell lymphoma. MLN4924, an E1 inhibitor of the Nedd8-activating enzyme (NAE) is currently in clinical oncology trials (Soucy, T. A., et al., 2009, An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer, Nature 458:732-736; Soucy, T. A., et al., 2009, Targeting NEDD8-activated cullin-RING ligases for the treatment of cancer, Clin Cancer Res 15:3912-3916) and numerous other targets within the ubiquitin/protein homeostasis arena are of interest for oncology (Nalepa, G., et al., 2006, Drug discovery in the ubiquitin-proteasome system, Nat Rev Drug Discov 5:596-613). Preclinical studies with PYZD-4409, a UAE inhibitor, showed that it induced cell death in both leukemia and myeloma cell lines and demonstrated anti-tumor activity in a mouse acute myeloid leukemia (AML model). (Xu, W. G., et al., 2010, The ubiquitin-activating enzyme E1 as a therapeutic target for the treatment of leukemia and multiple myeloma, Blood, 115:2251-59). Thus, UAE represents a novel protein homeostasis target opportunity for the treatment of cancer.
It is believed that UAE inhibitors would also be applicable for the treatment of other diseases and conditions outside of oncology due to the vast role of ubiquitin in cellular process; for example, proteasome inhibitors, which like UAE inhibitors alter cellular protein homeostasis, show promise for the treatment of antibody mediated transplant rejection (Woodle, E. S., et al., 2011, Proteasome inhibitor treatment of antibody-mediated allograft rejection, Curr Opin Organ Transplant 16:434-438), ischemic brain injury, infection, and autoimmune disorders (Kisselev, A. F., et al., 2012, Proteasome inhibitors: an expanding army attacking a unique target, Chem Biol 19:99-115). Ubiquitin-dependent signaling and degradation are important for the activation of pro-inflammatory pathways such as the NF-kB pathway implicating UAE inhibitors as potential anti-inflammatory agents Wertz, I. E., and Dixit, V. M., 2010, Signaling to NF-kappaB: regulation by ubiquitination, Cold Spring Harb Perspect Biol, 2:a003350).